This invention relates to a multiwell test apparatus which is utilized for promoting fluid interactions such as by growing cells in a nutrient medium within the wells. More particularly, this invention relates to such a multiwell filter plate of a multiwell test apparatus which permits adding or removing liquid from the feeding plate of a multiwell test apparatus without disturbing a material such as cells within the wells and two plates, one for growing the cells and the other for the assaying of the cells.
At the present time, multiwell test apparatus for testing samples include a multiwell filter plate, a single well feeding plate, a multiwell receiver plate and a lid. The wells of the multiwell filter plate are formed of a tubular member with an open end to which is attached a membrane such as a microporous membrane. The tubular members can be inserted into a single well feeding plate containing a nutrient medium so that cells in the wells can be attached to the membrane and grown thereon. The cells are fed as nutrients pass from the nutrient medium through the membrane and to the cells at a rate controlled by the concentration gradient of nutrients from the medium to the cells. The nutrient medium in the feed tray is periodically replenished to maintain cell growth. It is desirable to effect replenishment of the nutrient medium quickly and in a manner which avoids damage to the membranes and the cells.
After the desired level of cell growth on the membranes of the wells has been attained, the multiwell filter plate can be utilized in conventional assay methods. These assay methods generally are effected by positioning the membranes and cells on the multiwell filter plate into the wells of the multiwell receiver plate positioned below the multiwell filter plate having the same number of wells in register with the wells of the filter plate. The wells of the multiwell receiver plate contain a liquid composition to be assayed. The composition to be assayed diffuses into the cells and then through the membrane. The resultant liquid products within the wells of the multiwell filter plate or in the wells of the multiwell receiver plate then are assayed to determine the capability of the composition being assayed to permeate the cell barrier.
An important component in the drug discovery and development process is the determination of the oral absorption and bioavailability of new compounds. In order to perform this evaluation in a cost effective, high throughput and sensitive assay, it is ideal to use an in vitro device with a multitude of wells, containing cells and a small amount of assay material and automation. Classically, the determination of in vitro oral absorption characteristics is performed using a defined epithelium cell line and measuring the apparent transport rate of the drug across a monolayer of the cells. More recently it is possible to rank/order the passive transport rate of potential drug candidates using an artificial membrane barrier. The values generated from these in vitro experiments are valuable methods for screening the most likely successful drug candidates long before the oral absorption rate are validated by in vivo measurements. A typical experiment for determining the drug absorption characteristics of a known or unknown chemical compound is performed as follows. The multiwell device is seeded with epithelium cells on top of the filter in a defined medium. The same medium is also added to the single well feeding plate, located below and in fluid contact with the device containing the cells. The cells are allowed to proliferate and differentiate over a number of days. The nutrient medium is periodically replaced with fresh medium to replenish exhausted nutrients and remove waste and dead cells. At the end of a growing time, the cells and multiwell device are gently washed with an isotonic buffer to remove protein and residual nutrient medium. At this time, the multiwell filter plate is transferred to the multiwell receiver plate and the chemicals to be assayed are introduced to either the compartment above the cell layer or below the cells and filter support in the multiwell receiver tray. The opposing chamber is filled with drug free buffer and the multiwell device is incubated for some period of time, typically at 37 degrees Centigrade with shaking. Samples then are removed from either compartment for assay. If multiple time points are desired, multiple time-spaced samples are taken from either compartment, and buffer is added to the compartment to replace the sample volume. It is important that the membrane or cell layer not be disturbed during sample removal or buffer addition. The amount of drug/chemical that is transported across the cell barrier can be determined by a variety of analytical methods, but typically is determined using LC-MS/MS (Liquid Chromatography-Mass Spectrometry-Mass Spectrometry).
After the cells have been satisfactorily grown and the single well feeding plate is to be replaced by the multiwell receiver plate, it is desirable to minimize transport of the nutrient medium to the multiwell receiver plate thereby to minimize dilution of the composition being assayed. Thus, it is desirable to remove any droplets of nutrient medium retained on the lower surfaces of the membranes after the multiwell filter plate is removed from the nutrient medium in the single well feeding plate.
Also, it would be desirable to provide a multiwell filter plate of a multiwell test apparatus which facilitates liquid addition or removal from the wells of the multiwell filter plate in a manner which preserves the integrity of the membranes and when present, cells on the membrane. In addition, it would be desirable to provide such a multiwell filter plate which can be utilized in conjunction with a multiwell receiver plate subsequent for use in conjunction with the single well feeding plate.
Also it would be desirable to provide a multiwell test apparatus with a feed plate that has various means for the provision or removal of liquids in the feed plate without disturbing the cells and which minimizes the sloshing of the liquid in the feed tray during handling.